M.D. Mayan

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim of this study was to investigate if chondrocytes from human articular cartilage express gap junction proteins called connexins (Cxs). We show that human chondrocytes in tissue express Cx43, Cx45, Cx32, and Cx46. We also find that primary chondrocytes from adults retain the capacity to form functional voltage-dependent gap junctions. Immunohistochemistry experiments in cartilage from OA patients revealed significantly elevated levels of Cx43 and Cx45 in the superficial zone and down through the next approximately 1000 μm of tissue. These zones corresponded with regions damaged in OA that also had high levels of proliferative cell nuclear antigen. An increased number of Cxs may help explain the increased proliferation of cells in clusters that finally lead to tissue homeostasis loss. Conversely, high levels of Cxs in OA cartilage reflect the increased number of adjacent cells in clusters that are able to interact directly by gap junctions as compared with hemichannels on single cells in normal cartilage. Our data provide strong evidence that OA patients have a loss of the usual ordered distribution of Cxs in the damaged zones and that the reductions in Cx43 levels are accompanied by the loss of correct Cx localization in the nondamaged areas.

Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis

Objective This study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels. Methods Frozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids. Results Microscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150 µm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and β-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12 min after injection. Glucose was homogeneously distributed within 22 and 35 min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes. Conclusions This study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell–cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue.

Proteomic Analysis of Connexin 43 Reveals Novel Interactors Related to Osteoarthritis

We have previously reported that articular chondrocytes in tissue contain long cytoplasmic arms that physically connect two distant cells. Cell-to-cell communication occurs through connexin channels termed Gap Junction (GJ) channels, which achieve direct cellular communication by allowing the intercellular exchange of ions, small RNAs, nutrients, and second messengers. The Cx43 protein is overexpressed in several human diseases and inflammation processes and in articular cartilage from patients with osteoarthritis (OA). An increase in the level of Cx43 is known to alter gene expression, cell signaling, growth, and cell proliferation. The interaction of proteins with the C-terminal tail of connexin 43 (Cx43) directly modulates GJ-dependent and -independent functions. Here, we describe the isolation of Cx43 complexes using mild extraction conditions and immunoaffinity purification. Cx43 complexes were extracted from human primary articular chondrocytes isolated from healthy donors and patients with OA. The proteomic content of the native complexes was determined using LC-MS/MS, and protein associations with Cx43 were validated using Western blot and immunolocalization experiments. We identified >100 Cx43-associated proteins including previously uncharacterized proteins related to nucleolar functions, RNA transport, and translation. We also identified several proteins involved in human diseases, cartilage structure, and OA as novel functional Cx43 interactors, which emphasized the importance of Cx43 in the normal physiology and structural and functional integrity of chondrocytes and articular cartilage. Gene Ontology (GO) terms of the proteins identified in the OA samples showed an enrichment of Cx43-interactors related to cell adhesion, calmodulin binding, the nucleolus, and the cytoskeleton in OA samples compared with healthy samples. However, the mitochondrial proteins SOD2 and ATP5J2 were identified only in samples from healthy donors. The identification of Cx43 interactors will provide clues to the functions of Cx43 in human cells and its roles in the development of several diseases, including OA.

Recruitment of RNA molecules by connexin RNA-binding motifs: Implication in RNA and DNA transport through microvesicles and exosomes

Connexins (Cxs) are integral membrane proteins that form high-conductance plasma membrane channels, allowing communication from cell to cell (via gap junctions) and from cells to the extracellular environment (via hemichannels). Initially described for their role in joining excitable cells (nerve and muscle), gap junctions (GJs) are found between virtually all cells in solid tissues and are essential for functional coordination by enabling the direct transfer of small signalling molecules, metabolites, ions, and electrical signals from cell to cell. Several studies have revealed diverse channel-independent functions of Cxs, which include the control of cell growth and tumourigenicity. Connexin43 (Cx43) is the most widespread Cx in the human body. The myriad roles of Cx43 and its implication in the development of disorders such as cancer, inflammation, osteoarthritis and Alzheimers disease have given rise to many novel questions. Several RNA- and DNA-binding motifs were predicted in the Cx43 and Cx26 sequences using different computational methods. This review provides insights into new, ground-breaking functions of Cxs, highlighting important areas for future work such as transfer of genetic information through extracellular vesicles. We discuss the implication of potential RNA- and DNA-binding domains in the Cx43 and Cx26 sequences in the cellular communication and control of signalling pathways.

The C-terminal domain of connexin43 modulates cartilage structure via chondrocyte phenotypic changes

Chondrocytes in cartilage and bone cells population express connexin43 (Cx43) and gap junction intercellular communication (GJIC) is essential to synchronize cells for coordinated electrical, mechanical, metabolic and chemical communication in both tissues. Reduced Cx43 connectivity decreases chondrocyte differentiation and defective Cx43 causes skeletal defects. The carboxy terminal domain (CTD) of Cx43 is located in the cytoplasmic side and is key for protein functions. Here we demonstrated that chondrocytes from the CTD-deficient mice, K258stop/Cx43KO and K258stop/K258stop, have reduced GJIC, increased rates of proliferation and reduced expression of collagen type II and proteoglycans. We observed that CTD-truncated mice were significantly smaller in size. Together these results demonstrated that the deletion of the CTD negatively impacts cartilage structure and normal chondrocyte phenotype. These findings suggest that the proteolytic cleavage of the CTD under pathological conditions, such as under the activation of metalloproteinases during tissue injury or inflammation, may account for the deleterious effects of Cx43 in cartilage and bone disorders such as osteoarthritis.

Cartilage regeneration and ageing: Targeting cellular plasticity in osteoarthritis

Ageing processes play a major contributing role for the development of Osteoarthritis (OA). This prototypic degenerative condition of ageing is the most common form of arthritis and is accompanied by a general decline, chronic pain and mobility deficits. The disease is primarily characterized by articular cartilage degradation, followed by subchondral bone thickening, osteophyte formation, synovial inflammation and joint degeneration. In the early stages, osteoarthritic chondrocytes undergo phenotypic changes that increase cell proliferation and cluster formation and enhance the production of matrix-remodelling enzymes. In fact, chondrocytes exhibit differentiation plasticity and undergo phenotypic changes during the healing process. Current studies are focusing on unravelling whether OA is a consequence of an abnormal wound healing response. Recent investigations suggest that alterations in different proteins, such as TGF-ß/BMPs, NF-Kß, Wnt, and Cx43, or SASP factors involved in signalling pathways in wound healing response, could be directly implicated in the initiation of OA. Several findings suggest that osteoarthritic chondrocytes remain in an immature state expressing stemness-associated cell surface markers. In fact, the efficacy of new disease-modifying OA drugs that promote chondrogenic differentiation in animal models indicates that this may be a drug-sensible state. In this review, we highlight the current knowledge regarding cellular plasticity in chondrocytes and OA. A better comprehension of the mechanisms involved in these processes may enable us to understand the molecular pathways that promote abnormal repair and cartilage degradation in OA. This understanding would be advantageous in identifying novel targets and designing therapies to promote effective cartilage repair and successful joint ageing by preventing functional limitations and disability.

Intercellular communication via gap junction channels between chondrocytes and bone cells

Cell-to-cell communication between bone, cartilage and the synovial membrane is not fully understood and it is only attributed to the diffusion of substances through the extracellular space or synovial fluid. In this study, we found for the first time that primary bone cells (BCs) including osteocytes, synovial cells (SCs) and chondrocytes (CHs) are able to establish cellular contacts and to couple through gap junction (GJ) channels with connexin43 (Cx43) being dominant. Transwell co-culture and identification by mass spectrometry revealed the exchange of essential amino acids, peptides and proteins including calnexin, calreticulin or CD44 antigen between contacting SCs, BCs and CHs. These results reveal that CHs, SCs and BCs are able to establish intercellular connections and to communicate through GJ channels, which provide a selective signalling route by the direct exchange of potent signalling molecules and metabolites.

FRI0031 Targeting Sialic Acid-Modified Receptors as A Potential Therapy for Osteoarthritis

Background Osteoarthritis (OA) is one of the most common diseases worldwide. Its prevalence and severity increase with age, but treatments only provide symptomatic relief. The articular cartilage consists of a collagen-proteoglycan matrix containing highly glycosylated proteins synthesized by chondrocytes. A notable shift from glycoproteins containing a-2,6-linked sialic acids to those containing a-2,3-linked sialic acids has been associated with progressive cartilage degeneration and with the onset of disorders such as rheumatoid arthritis (RA) and OA1–3. However, the pathophysiology of a-2,3-sialylation in cartilage has not yet been elucidated. Objectives Lectins recognize specific terminal aspects of glycan chains and the Maackia amurensis seed lectin (MASL) is a plant lectin that can bind to sialylated glycoproteins. Here, we study the effects of the lectin MASL on chondrocytes and cartilage integrity from healthy donors, OA patients and animal models of arthritis. Methods Cell viability, cell adhesion and growth were performed using commercial kits. Reactive oxygen species (ROS) levels were measured by DCFH-DA and by Flow Cytometry. Gene expression was analyzed by quantitative real-time PCR. Staining methods were used to study cartilage integrity. Oligomycin and LPS were used to induce cartilage degeneration in vitro and in vivo (human cartilage and mouse model, Mus musculus BALC/c). Results he expression of the a-2,3-sialylated transmembrane mucin receptor podoplanin (PDPN) is induced in cartilage from osteoarthritic patients. Co-immunofluorescence technique showed that MASL can be used to target PDPN. Nanomolar concentrations of MASL protected primary chondrocytes and prevented cartilage breakdown in human tissue from OA patients ex vivo and in an animal model of OA initiated by ROS, inflammatory cytokines, and metalloproteinases. Besides, the increased levels of the α-2,3 sialyltransferase isoforms and the corresponding increase in the levels of a-2,3-sialylated glycoproteins in osteoarthritic chondrocytes may shed mechanistic light on the pathophysiology of OA. These findings based on various experimental models of arthritis reveal that specific lectins that target a-2,3-sialylated transmembrane receptors, such as PDPN on chondrocytes, may effectively inhibit cartilage destruction in the face of various arthritic insults. We also provide a three-dimensional molecular model for such an interaction. Conclusions The ability of MASL to target a-2,3-sialylated glycoproteins, such as PDPN, and to protect chondrocytes from insults leading to cartilage degradation might offer further possibilities for therapeutic interventions and novel arthritis treatments that may include the regulation of sialylation during acute disease stages. References Toegel, S. et al. Arthritis Res Ther 15, R147. (2013). Toegel, S. et al. Osteoarthritis Cartilage 18, 240–248. (2010). Toegel, S. et al. In Vitro Cell Dev Biol Anim 45, 351–360. (2009). Disclosure of Interest None declared

FRI0024 Olive-Derived Oleuropein as A Potential Treatment for Bone and Cartilage Age-Related Disorders

Background Oleuropein is the major glycoside component in olives and is well known to have several beneficial effects on human health [1]. However, the mechanisms associated with its potential pharmacological properties are not clear yet. Human bone marrow mesenchymal stem cells (hMSCs) exhibit an age-dependent reduction in osteogenesis and an increased propensity toward adipocyte differentiation. This switch has been associated with different bone disorders characterized by reduced bone formation and increased bone marrow fat accumulation [2,3]. Connexin 43 (Cx43) is an integral membrane protein that forms gap junction channels (GJs) and it is implicated in multiple cellular functions including cellular differentiation and control of bone and cartilage remodelling. Objectives The aim of this study was to test if oleuropein could act as an adipogenic suppressor in order to promote cartilage and bone regeneration through a Cx43-dependent mechanism. Methods Human mesenchymal stem cells were obtained from bone marrow donors. Cells were grown in α-minimum essential medium (MEM) and differentiation was initiated before achieving confluence with specific differentiation media in the presence of different concentrations of oleuropein. Adipogenesis and osteogenesis was held in culture chambers for 21 days; chondrogenesis was performed as a micromass culture for 30 days. Cellular differentiation was evaluated using histological stains: Oil Red O for adipogenesis; Alizarin Red for osteogenesis; Toluidine Blue and Safranin O/Fast Green for chondrogenesis. Scrape loading assays and lucifer yellow were used to study the cellular communication through gap junction (GJ) and hemichannels formed by Cx43. Western-blot and immunohistochemistry (IHC) assays were performed to study the levels of Cx43 protein. Results hMSCs treated with oleuropein showed a two-folded decrease in adipogenic differentiation, while osteogenesis was significantly increased. Micromasses cultured in chondrogenic medium using 10 μM of oleuropein showed a more intense toluidine blue and Safranin O stain, suggesting an increase in chondrogenesis. Real-Time qPCR, western blot analysis and scrape loading assays showed changes in the levels of Cx43 and dye transference through GJ channels when the hMSCs were grown in the presence of oleuropein. Conclusions Our results suggest that oleuropein via Cx43 and GJ intercellular communication increases the propensity towards osteogenesis and chondrogenesis, reducing adipocyte differentiation. Our preliminary assay indicates that oleuropein may represent a potential therapeutic target for cartilage and bone age-related disorders such as osteoarthritis in order to promote cartilage and bone regeneration. References Barbaro B et al. Int J Mol Sci (2014). 15 (10): 18508–18524. Lee-Huang S et al. Open Conf Proc J (2013). 4: 113–124. Santiago-Mora R et al. Osteoporos Int (2011). 22: 675–684. Disclosure of Interest None declared

AB0072 Recruitment of RNA Targets by Connexin 43 RNA-Binding Motifs: Novel Mechanisms of Cellular Communication

Background Connexins (Cxs) are integral membrane proteins that form plasma membrane channels, allowing cell-matrix and cell to cell communication. Initially described in joining excitable cells (nerve and muscle), gap junctions (GJs) are found joining virtually all cells in solid tissues and are essential for the functional co-ordination of organs by enabling direct transfer of small signalling molecules, metabolites, ions, and electrical signals. Several studies have revealed diverse channel-independent functions of Cxs, including control of cell growth and tumorigenicity. Chondrocytes from osteoarthritic cartilage have increased expression of connexin 43 (Cx43). The myriad roles of Cx43 and its implication in the development of diseases such as cancer, osteoarthritis or alzheimer have rise to many novel questions. Objectives The aim of this study was to investigate possible RNA binding domains in the Cx43 and Cx26 sequences in order to study if cells are able to exchange molecules of RNA anchored to Cx43/Cx26 sequences present in exosomes or vesicles. Methods Amino acids sequences from Cx43 and Connexin 26 (Cx26) were obtained from the Protein Database (NCBI) and the Cx26 structure from the Protein Data Bank (RCSB) [1]. Protein sequences from both Cxs were aligned and compared with Protein Blast and Lalign servers. Analysis of RNA-binding propensity of Cx43 and Cx26 sequences was evaluated with three computational methods with the high predicting accuracy [2] aaRNA, Bind+ and Pprint. Results Protein sequences were highly conserved between Cx43 and Cx26 with the exception of the the C-terminal end (CTD). The combination of the scores obtained from the three predictors showed four domains that potentially interact with RNA sequences. Two of these domains include the intracellular loop (ICL) and the CTD. Conclusions Our results suggest that Cx43 and Cx26 are able to interact with RNA targets including siRNA and mRNA. These interactions would have a range of potential implications for cellular communication and control of signalling pathways. References Maeda S et al. Nature 458: 597–602. Nagarajan R et al. PLos ONE 9 (3): e91140. Disclosure of Interest None declared